WO2017119665A1 - Composition for preventing and treating influenza viruses comprising lactic acid bacteria as active ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing and treating influenza viruses comprising lactic acid bacteria as active ingredients, wherein the deposited strain L. plantarum of the present invention can be used as an effective antiviral probiotic microorganism.

Description

Influenza virus prevention and treatment composition comprising lactic acid bacteria as an active ingredient

The present invention relates to a composition for preventing and treating influenza viruses comprising lactic acid bacteria as an active ingredient.

Influenza, which causes human influenza, has occurred every year since the Spanish flu in 1919, causing great damage. In addition, avian influenza, which infects chickens, ducks, and migratory birds, has been damaging the human and poultry industries worldwide for many years. Influenza is susceptible to strains. In 2009, a new strain of influenza occurred, affecting approximately 480,000 people worldwide, of which 6,200 died (WHO). In 2010, 79 people died of swine flu in India. It is also likely that new influenza resistant to antiviral drugs will emerge. Influenza viruses are particles with a diameter of 0.1 nm, have epidermis, and have RNA inside.

The influenza viruses are classified into types A, B, and C, and the epidemic diseases are type A and B. The epidermis of the influenza virus protrudes two kinds of glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and has 9 segments of RNA inside. Hemagglutinin (HA) and neuraminidase (NA) on the surface of the influenza virus cause mutations frequently, and new strains are frequently appearing.

Influenza viruses invade the nose and mouth of animals such as humans, pigs, and chickens, and the hemagglutinin (HA) of the epidermis binds to sialic acid residues in mucosal epithelial cells of the upper airways, and invades RNA into cells. Influenza virus RNA is replicated and neuraminidase (NA) breaks down sialic acid in the host cell membrane to release the progeny influenza virus as budding. Amantadine, rimantadine, oseltamivir phophate (Tamiflu), zanamivir (Relenza, Relenza) are known to suppress the infection of these influenza viruses. . Amantadine and rimantadine block the ion channel function of the M2 protein of the influenza virus, and some influenza have no inhibitory effect, make a variant virus, and have severe side effects such as anxiety, dullness, and seizures. Oseltamivir phosphate (Tamiflu) and zanamivir (Relenza) are inhibitors of the neuraminidase enzyme activity in the epidermis of influenza viruses. However, this drug also has side effects such as hypersensitivity, mental neuropathy, and indigestion, and is still in limited use.

In such a situation, there is a need for an anti-influenza development plan that effectively prevents or treats influenza and can be used safely.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide an influenza treatment and prevention agent that can be effectively protected from influenza and that can be used safely.

In order to achieve the above object, the present invention provides a composition having a protective effect against influenza virus comprising a Lactobacillus plantarum strain as an active ingredient.

In one embodiment of the present invention, the Lactobacillus plantarum is preferably an accession number KCTC12946BP, but is not limited thereto.

In another embodiment of the present invention, the composition is preferably for nasal or oral administration, but is not limited thereto.

In another embodiment of the present invention, the composition is preferably a pharmaceutical, food or feed composition, but is not limited thereto.

The protective effect against the antiviral of pharmaceutical compositions, food compositions or feed compositions comprising Lactobacillus plantarum or fragments thereof having accession number KCTC12946BP of the present invention was very good.

The herbal preparations of the pharmaceutical compositions according to the invention can be made in a general manner in the art. Suitable solid or liquid herbal formulations are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions or emulsions, and for their preparation, carrier materials, explosives, binders, coatings, expanding agents, lubricants Conventional means such as, flavors, sweeteners and solution mediators are used. As auxiliary substances here animal and vegetable oils, such as magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, cellulose and derivatives, cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol And solvents such as sterile water and monohydric or polyhydric alcohols such as glycerin. The pharmaceutical composition according to the present invention is prepared by mixing the lactic acid bacterium according to the present invention in a predetermined amount with a pharmaceutically suitable and physiologically resistant carrier and optionally additional suitable activity, additions or auxiliary substances, and formulated in a desired dosage form. can do.

The composition may contain or consist of 0.1 to 95% by weight carrier and 5 to 99.9% by weight lyophilized lactic acid bacteria relative to the total amount of cells and carriers.

The lactic acid bacteria of the present invention can be used as direct inoculation products. Thus, bacteria can be added to foods, or pharmaceutical compositions, without any prior processing steps. It is preferred that the bacteria of the invention are cryopreserved in their own fermentation supernatants. This facilitates the production of the product and thus saves time and money.

10 ^ 2 to 10 ^ 15, preferably 10 ^ 6 or 10 ^ for food compositions, animal feed, pharmaceutical compositions in absolute value or for 100 g of food, animal feed, or pharmaceutical compositions containing cells It is preferred to contain 8 to 10 ^ 12, lactic acid bacterial cells or fragments thereof.

"Fragment of the microorganism of the present invention" encompasses any part of the cells of the microorganism of the present invention. Preferably, the fragments are membrane fragments obtained from membrane preparations. Membrane preparations of microorganisms belonging to the genus Lactobacillus are described by methods known in the art, for example in Rollan et al., Int. J. Food Microbiol. 70 (2001), 303-301, Matsuguchi et al., Clin. Diagn. Lab. Immunol. 10 (2003), 259-266 or Stentz et al., Appl. Environ. Microbiol. 66 (2000), 4272-4278 or Varmanen et al., J. Bacteriology 182 (2000), 146-154.

Hereinafter, the present invention will be described.

We isolated several Lactic acid bacteria strains (hereinafter referred to as 'LABS') from fermented foods (kimchi, soybean paste) and mushrooms, and antiviral of all LABS against influenza virus influenza virus A / NWS / 33. Activity was screened and finally the protective effect of the top Lactobacillus candidates by two routes of administration (nasal and oral) against influenza virus A / Korea / 01/2009 (H1N1) in mice was analyzed.

We isolated 16 LABS; 4 Lactobacillus plantarum , 5 Lactobacillus brevis , 2 Pediococcus acidilactici , 2 Pediococcus pentosaceus , 1 Lactobacillus sakei, and 1 Lactobacillus cuvatus

In addition, mice were screened for their protective effect against influenza virus A / NWS / 33. L at the first screening . plantarum - 2, L. brevis -5, P. pentosaceus -1, P.pentosaceus -2, P. acidilactici -2, and L. sakei were 90%, 90%, 87.5%, 80%, 80% and 77.77, respectively. It was selected as an excellent probiotic candidate strain by yielding% survival. Three candidate strains were also selected to test the effect of in vivo protection by oral and nasal administration against influenza virus A / Korea / 01/2009 (H1N1) in mice.

Compared to oral administration, nasal administered L. plantarum- 2, L. sakei and L. brevis-5 increased protection against influenza virus infection. L. plantarum -2 showed 100% protection against influenza virus, followed by L. brevis -5 (90%) and L. sakei (80%), with significant weight loss with alleviated clinical symptoms Suppressed. These results indicate that L. plantarum can be developed as an effective antiviral probiotic microorganism.

L. plantarum -2 showing a 100% protective effect of the present invention was deposited on the microbial resource center (KCTC), 125, Gwahak-ro, Yuseong-gu, Daejeon, Korea, on November 12, 2015 under the accession number KCTC12946BP.

As can be seen from the present invention, the deposited strain L. plantarum of the present invention can be used as an effective antiviral probiotic microorganism.

1 (A) Lactobacillus plantarum strain (B) Lactobacillus brevis strain (C) Lactobacillus acidilactici strain, Lactobacillus pentosaceus strain, Lactobacillus sakei and Lactobacillus curvatus strains after inoculation of influenza virus A / NWS / 33 inoculation of mice,

Figure 2 is a (A) Lactobacillus plantarum strain (B) Lactobacillus brevis strain (C) Lactobacillus acidilactici strain, Lactobacillus pentosaceus strain, Lactobacillus sakei and Lactobacillus curvatus strains after inoculation of influenza virus A / NWS / 33 of the mice treated ,

3 is a graph of body weight change (A) and survival graphs after inoculation of influenza virus A / H1N1 in mice treated with L. plantarum- 2, L. sakei, and L. brevis- 5 separately by nasal and oral administration (B). ).

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: used for animals and viruses

Six-week old female specific pathogen-free (SPF) BALB / c mice (Orient Bio Laboratories, Seoul, Korea) weighed 18-20 g. All experiments were performed in accordance with the protocol with the approval of the Animal Experimental Ethics Committee of Konkuk University.

Influenza Virus A / NWS / 33 (H1N1) and Influenza Virus A / Korea / 01/2009 (H1N1) were grown in the lumen cavity of 11-day-old chicken embryos at 37 ^° C. for 2 days. Allantoic fluid was collected and stored at 70 ^° C. until use.

Viral titers were determined at 80% egg infective dose (EID ₈₀ ) in meninges. In summary, serial 10-fold diluted ureters were injected into embryonated eggs and the presence of the virus in each egg's ureter was determined based on the hemagglutinating ability of the virus. To make the virus adapted to mice, the virus was first isolated from mouse lungs, propagated in embryonated eggs / SPG eggs and re-inoculated into mice. In the challenge study, mice were anesthetized by intraperitoneal injection of Avertin (375 mg / kg) and then intranasally challenged with ₉₀ μL of 10 ^4.5 EID ₅₀ influenza virus.

Example  2: Isolation of Lactic Acid Bacteria from Korean Fermented Foods

Lactic acid bacteria were isolated from fermented Korean foods; Kimchi (Chinese cabbage, diced rice, and Dongchimi) and Doenjang from various parts of Korea. For LABS isolation, 1 ml fermentation soup was first added to 30 ml MRS broth and incubated at 37 ^° C. for 24 hours. One loop of cultured broth was streaked on Rogosa Agar plates and incubated at 37 ^° C. for 48 hours. Each grown colonies were further screened in MRS agar and incubated at 37 ^° C. for 24 hours. For the identification of LAB strains, single colonies isolated from each site were sequenced and LABs were identified by 16s rRNA gene sequencing. LABS isolated from fermented foods are included in Table 1.

	LAB strain
One	L. plantarum - 1
2	L. plantarum - 2
3	L. plantarum - 3
4	L. plantarum - 4
5	L. brevis - 1
6	L. brevis - 2
7	L. brevis - 3
8	L. brevis - 4
9	L. brevis - 5
10	L. brevis - 6
11	P. acidilactici -1
12	P. acidilactici -2
13	P. pentosaceus -1
14	P. pentosaceus -2
15	L. sakei
16	L. curvatus

Table 1 shows the different lactic acid bacteria (LABs) isolated.

Experimental Example

Influenza Virus A / NWS LABS protection against / 33

Several LABS were isolated from fermented foods and their protective effect was observed against influenza virus A / NWS / 33 in mice. Animals were assigned to 18 groups; 16 test groups, 1 positive and 1 negative group, 10 mice in each group for the first screening. Intranasal administration of each isolated strain at a concentration of 1 × 10 ⁸ cfu per mouse was performed for 6 days prior to the virus challenge. Positive and negative control groups were inoculated with PBS in the same form as the test group. After challenge with influenza virus A / NWS / 33 (10 ^4.5 / 90 μL / dose) on day 7, the clinical symptoms, mortality and body weight change were observed for 2 weeks to assess the protective effect of influenza virus in nasal-administered lactic acid bacteria. The pathogenicity index of the mice (mean score per mouse observed for 14 days) was 0 for normal, 1 for sick (fever, eyelid abnormalities, hair abnormalities), and 2 for death.

Protective Effect of Lactobacillus on Influenza Virus A / Korea / 01/2009 with Different Administration Routes

Three strains resistant to influenza virus A / NWS / 33 infection were selected for three weeks to further analyze the protective effect against influenza virus A / Korea / 01/2009 (H1N1). Animals were assigned to 8 experimental groups (n = 10 / group); 6 test groups, 1 positive control and 1 negative control. Mice were given nasal (90 ul) and oral (300 ul) administration of each LAB at a concentration of 1 × 10 ⁸ cfu / mouse for 8 days prior to viral challenge. Positive and negative controls were inoculated with PBS in the same form as the test group. Mice were challenged with influenza virus A / Korea / 01/2009 (H1N1) LD ₈₀ (10 ^4.5 / 90 μL / dose) on day 9. Clinical symptoms, mortality, and body weight changes were then observed for two weeks to assess the protective effect of influenza virus in nasal and orally administered lactic acid bacteria. The pathogenicity index of the mice (mean score per mouse observed for 14 days) was 0 for normal, 1 for sick (fever, eyelid abnormalities, hair abnormalities), and 2 for death.

The results of the Examples and Experimental Examples are as follows.

Anti-Influenza Effects of Different Lactic Acid Bacteria

Antiviral activity of each isolated strain against influenza virus A / NWS / 33 in mice was evaluated and divided into groups I, II, and III. Group I includes 4 test groups L. plantarum 1-4, group II includes 6 test groups L. brevis 1-6 and the third group 6 test groups; P. acidilactici 1-2, P. pentosaceus 1-2, L. sakei -1 and L. curvatus -1, each group contains 10 mice and has positive and negative controls. Nasal administration of each live Lactobacillus strain was continued for 6 days in all test groups and PBS was inoculated into the positive and negative controls. Challenge experiments were performed on day 7 and followed for two weeks to observe clinical symptoms and inhibitory effects of death. Body weight changes were recorded daily in mice after infection.

There was no apparent change in body weight change in the challenge-free mice (negative control), but the weight of the positive control and test groups decreased after inoculation of influenza virus A / NWS / 33. Body weight decreased from day 1 and decreased from L. plantarum -1, 2, 3, and 4 to 8, 10, 12, and 10 days of viral infection, and then restored to their original condition until 9, 11, 13, and 11, respectively ( 1A). In group II, body weights increased after 12, 10 and 8 days of infection in L. brevis- 1, 2-4, 5-6, respectively (FIG. 1B). On the other hand, in P. pentosaceus 1-2, L. sakei- 1, L. curvatus- 1, and P. acidilactici 1-2, weight loss increased 8 and 9 days after infection (FIG. 1C). Mean onset time of clinical symptoms was 3 days in all groups.

All mice died in the influenza virus inoculated group (positive control) and not in the sham inoculated group (negative control). In group I, L. plantarum- 2 exhibited the highest protection with a survival rate of 90% and less than 60% in other L. plantarum strains (FIG. 2A). L. brevis -5, with a pathogenicity index of 1.1 and 90% survival, showed the highest protective effect, followed by L. brevis -6, L. brevis -3, L. brevis -1, and L. brevis -2. Were 77.77%, 55.55%, 50%, 22.22% and 10%, respectively (FIG. 2B). On the other hand, in group III, P. pentosaceus -1 showed a survival rate of 87.5%, followed by P. pentosaceus -2, P. acidilactici -2, L. sakei , L. curvatus and P. acidilactici -1, respectively. Survival rates of 80%, 80%, 77.77%, 55.55% and 50% were shown (FIG. 2C). Among the group III strains, L. sakei had the lowest pathogenicity index of 1.2.

Protective Effect of Lactic Acid Bacteria with Different Routes of Administration

Three candidate strains; Three candidate strains with excellent antiviral effects against L. plantarum- 2, L. brevis- 5 and L. sakei having better antiviral effect against influenza virus A / NWS / 33; L. plantarum- 2, L. brevis- 5 and L. sakei were selected and evaluated for their protective effect against influenza virus A / Korea / 01/2009 (H1N1) in mice after oral and nasal administration of Lactobacillus strains. Mice were divided into 8 groups containing 10 animals in each group. G1-G3 was treated with L. plantarum- 2, L. brevis- 5 and L. sakei by nasal route, and G4-G6 by L oral route . treated with plantarum- 2, L. brevis- 5 and L. sakei , G7 (positive control) and G8 (negative control) were treated with PBS for 8 days and challenged with influenza virus A / H1N1. Body weight changes, clinical symptoms, and death inhibition effects were then observed for two weeks. Lactobacillus an intranasal treatment with the relaxed clinical symptoms was inhibited significant weight loss (Fig. 3A). The weight loss was increased by 10 days after infection in the L. plantarum and L. brevis -2 -5 intranasal administration, L It increased from 9th day in sakei .

Compared to orally administered Lactobacillus , nasal treated Lactobacillus showed a higher protective effect against influenza virus A / H1N1. L. plantarum - 2 showed 100% protective effect against influenza virus, followed by L. brevis- 5 (90%) and L. sakei (80%) (FIG. 3B). In contrast, in the oral dosing group, most mice died after challenge experiments in 2 who survived the L. sakei case. The mean onset time of clinical signs was 3 days in all groups except the negative control. In addition, the observed clinical symptom body temperature (fever), eyelid abnormalities, and hair abnormalities were better in the LAB treated group than in the positive control group. In particular, the pathogenicity index was lower at 1.0 in L. plantarum-2, 1.1 at L. brevis-5 and 1.2 at L. sakei than oral administration.

[Accession number]

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC12946BP

Deposit date: 20151112

Claims (4)

1. A composition having a protective effect against influenza virus comprising Lactobacillus plantarum strain as an active ingredient.
2. The composition of claim 1, wherein the Lactobacillus plantarum has a protective effect against influenza virus, characterized in that Accession No. KCTC12946BP.
3. The composition according to claim 1 or 2, wherein the composition has a protective effect against influenza virus, which is for nasal or oral administration.
4. According to claim 1 or 2, wherein the composition is a pharmaceutical, food or feed composition having a protective effect against influenza virus, characterized in that the composition.

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